Comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR

Heungsup Sung, DongEun Yong, Chang Seok Ki, Jae Seok Kim, Moon Woo Seong, Hyukmin Lee, Mi Na Kim

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P <0.0001). Conclusions: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Original languageEnglish
Pages (from-to)457-462
Number of pages6
JournalAnnals of laboratory medicine
Volume36
Issue number5
DOIs
Publication statusPublished - 2016 Sep 1

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Homogenization method
Transcription
Sputum
Nucleic Acids
Reverse Transcription
Acetylcysteine
Endopeptidase K
Phosphates
Deoxyribonuclease I
Polymerase Chain Reaction
RNA
Sodium Chloride
Coronavirus Infections
Middle East Respiratory Syndrome Coronavirus
Coronavirus
Mucus
sodium citrate
Korea
France

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

@article{09dcadff399844bdaa55f546b0b34892,
title = "Comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR",
abstract = "Background: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioM{\'e}rieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7{\%}) and four samples (13.3{\%}), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P <0.0001). Conclusions: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.",
author = "Heungsup Sung and DongEun Yong and Ki, {Chang Seok} and Kim, {Jae Seok} and Seong, {Moon Woo} and Hyukmin Lee and Kim, {Mi Na}",
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language = "English",
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Comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR. / Sung, Heungsup; Yong, DongEun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na.

In: Annals of laboratory medicine, Vol. 36, No. 5, 01.09.2016, p. 457-462.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR

AU - Sung, Heungsup

AU - Yong, DongEun

AU - Ki, Chang Seok

AU - Kim, Jae Seok

AU - Seong, Moon Woo

AU - Lee, Hyukmin

AU - Kim, Mi Na

PY - 2016/9/1

Y1 - 2016/9/1

N2 - Background: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P <0.0001). Conclusions: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

AB - Background: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P <0.0001). Conclusions: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

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